This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The most direct and intuitive method of relative peptide quantification is comparison of the peak areas of the same peptide specie across different experiments. However, the difficulty of determining peaks that belong to the same peptide specie comes from poor reproducibility of the peptide retention time via HPLC. Combined use of the in-house packed micro columns with a second dimension of chromatographic separation during MudPIT experiments reduces retention reproducibility even further. We propose a chromatographic approach that takes advantage of the high retention reproducibility of a large 1mm i.d. RP column and combines it with the sensitivity of the nano-spray ESI. Chromatographic elution is carried out via long single phase gradients using large capacity 1.0x30.0 mm RP column. The high linear velocity flow (2ml/min) is split post column where the sample is introduced into the instrument at the nano-spray regime of 250nl/min. This setup accounts for improved chromatographic reproducibility, and increased detection sensitivity that is similar to the capillary micro-column nano-ESI setup